Annexin VI-mediated loss of spectrin during coated pit budding is coupled to delivery of LDL to lysosomes.

Kamal A, Ying Y, Anderson RG - J. Cell Biol. (1998)

Bottom Line:
Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding.The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded.Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN.

ABSTRACTPreviously we reported that annexin VI is required for the budding of clathrin-coated pits from human fibroblast plasma membranes in vitro. Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding. Annexin VI-dependent budding is accompanied by the loss of approximately 50% of the spectrin from the membrane and is blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Incubation of fibroblasts in the presence of ALLN initially blocks the uptake of low density lipoprotein (LDL), but the cells recover after 1 h and internalize LDL with normal kinetics. The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded. ALLN-treated cells have twice as many coated pits and twofold more membrane clathrin, suggesting that new coated pits have assembled. Annexin VI is not required for the budding of these new coated pits and ALLN does not inhibit. Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN. These findings suggest that fibroblasts are able to make at least two types of coated pits, one of which requires the annexin VI-dependent activation of a cysteine protease to disconnect the clathrin lattice from the spectrin membrane cytoskeleton during the final stages of budding.

Figure 6: Budding (A) is no longer linked to spectrin loss (B) in membranes from ALLN-treated cells. (A) Attached membranes obtained from cells incubated in the presence of ALLN for 1 h were warmed to 37°C in the presence of buffer alone (bar 1) or warmed in the presence of cytosol (bars 2–4) containing the indicated additions. At the end of the incubation, the percent loss of clathrin was measured as described. Maximum clathrin value was 58,321 cpm/well with a background of 2,536. (B) Attached membranes obtained from cells incubated in the presence of ALLN for 1 h were either not treated (bar 2) or warmed to 37°C for 10 min in the presence of cytosol (bars 3 and 4) containing the indicated additions. At the end of the incubation, the membranes were assayed for the amount of spectrin as described. Background binding is shown in bar 1. In both experiments, each value is the average of triplicate measurements ± the standard deviation.

Mentions:
Coated pits that assembled in the presence of ALLN did not require annexin VI for budding in vitro (Fig. 6 A). We incubated cells in the presence of ALLN for 1 h and prepared membranes. There was little loss of clathrin when these membranes were warmed to 37°C in the presence of buffer (Fig. 6 A, bar 1). Replacing the buffer with inactive cytosol, however, resulted in a 60% loss of clathrin (Fig. 6 A, bar 2). The same amount of clathrin loss occurred regardless of whether annexin VI was added to the cytosol (Fig. 6 A, compare bar 3 with 2) or if ALLN was present in the complete budding mixture (Fig. 6 A, compare bar 4 with 3). Consistent with these results, membranes from ALLN-treated cells did not lose any spectrin during budding (Fig. 6 B, compare bars 3 and 4 with 2). Apparently cells overcome the effects of ALLN by assembling a new population of pits that are not functionally or structurally linked to the spectrin cytoskeleton.

Figure 6: Budding (A) is no longer linked to spectrin loss (B) in membranes from ALLN-treated cells. (A) Attached membranes obtained from cells incubated in the presence of ALLN for 1 h were warmed to 37°C in the presence of buffer alone (bar 1) or warmed in the presence of cytosol (bars 2–4) containing the indicated additions. At the end of the incubation, the percent loss of clathrin was measured as described. Maximum clathrin value was 58,321 cpm/well with a background of 2,536. (B) Attached membranes obtained from cells incubated in the presence of ALLN for 1 h were either not treated (bar 2) or warmed to 37°C for 10 min in the presence of cytosol (bars 3 and 4) containing the indicated additions. At the end of the incubation, the membranes were assayed for the amount of spectrin as described. Background binding is shown in bar 1. In both experiments, each value is the average of triplicate measurements ± the standard deviation.

Mentions:
Coated pits that assembled in the presence of ALLN did not require annexin VI for budding in vitro (Fig. 6 A). We incubated cells in the presence of ALLN for 1 h and prepared membranes. There was little loss of clathrin when these membranes were warmed to 37°C in the presence of buffer (Fig. 6 A, bar 1). Replacing the buffer with inactive cytosol, however, resulted in a 60% loss of clathrin (Fig. 6 A, bar 2). The same amount of clathrin loss occurred regardless of whether annexin VI was added to the cytosol (Fig. 6 A, compare bar 3 with 2) or if ALLN was present in the complete budding mixture (Fig. 6 A, compare bar 4 with 3). Consistent with these results, membranes from ALLN-treated cells did not lose any spectrin during budding (Fig. 6 B, compare bars 3 and 4 with 2). Apparently cells overcome the effects of ALLN by assembling a new population of pits that are not functionally or structurally linked to the spectrin cytoskeleton.

Bottom Line:
Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding.The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded.Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN.

ABSTRACTPreviously we reported that annexin VI is required for the budding of clathrin-coated pits from human fibroblast plasma membranes in vitro. Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding. Annexin VI-dependent budding is accompanied by the loss of approximately 50% of the spectrin from the membrane and is blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Incubation of fibroblasts in the presence of ALLN initially blocks the uptake of low density lipoprotein (LDL), but the cells recover after 1 h and internalize LDL with normal kinetics. The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded. ALLN-treated cells have twice as many coated pits and twofold more membrane clathrin, suggesting that new coated pits have assembled. Annexin VI is not required for the budding of these new coated pits and ALLN does not inhibit. Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN. These findings suggest that fibroblasts are able to make at least two types of coated pits, one of which requires the annexin VI-dependent activation of a cysteine protease to disconnect the clathrin lattice from the spectrin membrane cytoskeleton during the final stages of budding.